The cornea is a transparent organ at the anterior of the eye which is critical to visual acuity. Corneal stroma and endothelia layers are derived from neural crest cells (NCCs) of the optic migratory stream. The fate of NCCs in their target tissue is influenced by their position along the rostro-caudal axis of the neural tube, their position within the migratory stream once they leave the neural tube, and induction from various signals within the microenvironments of their target tissues. Many of these inputs overlap in time;as such NCC specification likely relies on the integration of these influences. Dissecting the cellular events that shape the cornea in real time will provide tremendous insight into how the cornea is sculpted into a functional visual organ. I will employ 4D confocal imaging and photoconvertible fluorescent protein technology to observe these events, which will provide a more complete picture of corneal development. In order to track and observe subpopulations of NCCs, I will label the optic migratory stream with the KikGR photoconvertible fluorescent protein. Using this technology I will distinctly label subpopulations of the optic stream or their derivatives in the periocular region and track their fate as they invade the cornea. In addition, I will determine the response of each subpopulation to the cell guidance molecule Semaphorin3A (Sema3A) that has been shown to influence the ocular fate of NCCs. This proposal will undertake the following aims: 1: Define sub-populations of NCCs within the optic migratory stream and periocular region. 2: Define the cellular response of NCC-derived periocular cells to lens-derived signaling during cornea development by in vivo time-lapse video microscopy. 3: Determine whether lens-proximal and lens-distal NCC-derived periocular subpopulations exhibit differential migratory behavior in response to Sema3A signaling in vitro. These aims will fill gaps in our knowledge of how cells from the optic migratory stream acquire their positional identity and developmental program to give rise to corneal tissues. These findings will provide a jumping-off point for understanding the integration of multiple inputs to individual cells, which drive organogenesis in other systems. These aims are related but independent and each address an aspect of optic NCC progression toward a corneal fate. Findings from this study will shed light on the integration of multiple inputs and cellular behaviors during differentiation of NCC populations and will have important impacts on our understanding of congenital corneal defects. In addition, results from this study may have further reaching implications for migration of other NCC populations and invasive cell behaviors, leading to new information about dysregulation of these processes which can lead to craniofacial birth defects and some cancers. PUBLIC HEALTH RELEVANCE: The cornea is a transparent, avascular, and highly innervated tissue occupying the anterior position of the eye;proper development of this organ is critical to vision, and aberrant development can result in loss of visual acuity or blindness. The corneal stroma and endothelial layers are comprised of cells derived from the neural crest (NCCs), a transient migratory population that originates in the neural tube and gives rise to many organs, including the cornea. The optic stream of NCCs is known to contribute to the cornea, but it is not clear how or when the NCCs are specified toward their ultimate fate, a key event in cornea development. Here I propose to bridge this knowledge gap using real time 4D confocal imaging and photoconvertible fluorescent proteins to identify subpopulations of NCCs and their derivatives as they relate to corneal fate.